Aim 1: Identify critical signaling nodes that integrate RTK and hypoxia signaling pathways in RCC and, where relevant, in other GU malignancies, using HGF/Met as a model system. Progress in the development of prognostic markers of aggressiveness in clear cell renal cell carcinoma (RCC) has accelerated following new insights in molecular pathogenesis afforded by The Cancer Genome Atlas (TCGA) project for this disease. Although pathologic stage and grade are the best available tools at present for stratifying the risk of progression and survival, a significant proportion of cases have a clinical course that does not correlate with predictions. Using information gleaned the TCGA project and database, as well as our own in vitro models of aggressiveness in clear cell RCC, we have performed micro-array mRNA expression profiling analyses designed to identify critical nodes integrating RTK and hypoxia signaling networks. As reported previously, genes regulating cell cycle and division as well as cell-cell interactions were found to be modulated by VHL. However, further analysis using reverse modulation between the three different cell lines confirmed that some of these genes may also regulate branching morphogenesis and cell invasion. Surprisingly, the set of modulated genes also comprised multiple candidates derived from the BRCA/Fanconi anemia pathway, which establish a functional link between the latter and the VHL pathway that coordinates aggressive tumor activity and increased motility in clear cell RCC. We continue to validate of this set of genes using cellular and molecular biological experimentation with appropriately engineered model systems as well tumor-derived cell lines. As yet unpublished results indicate that isoforms of the full length protein encoded by the BARD1 gene are present in a variety of solid tumor-derived cell lines but not in normal cell lines derived from the same tissues/organs. These BARD1 protein isoforms appear to lack regulatory domains and their absence may contribute to the enhanced invasiveness of the tumor-derived lines. These truncated BARD1 isoforms are candidates for outcome and treatment response prognostication, and potential targets for new therapeutic strategies. Aim 3: Identify and develop selective natural product inhibitors of HIF2-alpha as therapeutic candidates for the treatment of clear cell RCC. Normoxic stabilization of HIF1-alpha alone, while capable of mimicking some aspects of VHL loss, are not sufficient to reproduce tumorigenesis and in fact, there is evidence that it acts as a tumor suppressor. HIF2-alpha, in contrast, is consistently cast as an oncoprotein. To isolate compounds that selectively modulate HIF2-alpha for use as research tools and drug development leads, a cell-based high throughput screening assay of the NCI Natural Products Repository was developed in collaboration with Drs. Tawnya McKee and James McMahon of CCR's Molecular Targets Program (MTP). A series of plasmids were engineered containing tandem copies of the minimal hypoxia response element of the VEGFA gene promoter upstream of a Luc reporter. These plasmids were transfected into the VHL- and HIF1-alpha-negative ccRCC cell line 786-0 and derived cell lines were optimized for use in high-throughput detection systems in the MTL. Secondary counter screens were included for global transcriptional repression and cell toxicity. Leads from Natural Products Repository screening were chromatographically separated into component structures yielding 40 pure compounds with micromolar or submicromolar IC50 values, 80% inhibition and 10% cell toxicity. Lead compounds have been further characterized for HIF1-alpha and HIF2-alpha selectivity using clear cell RCC-derived cell lines engineered to express either factor alone or both. Cells were exposed to lead compounds and modulation of a panel of HIF gene targets with known resposiveness to either factor or both was measured by qRT-PCR. Three lead compounds were found to signicantly reduce VEGF gene transcription and protein secretion, and selectively downregulate HIF2-alpha target genes. Using the Peloton Therapeutics drug candidate PT2385 now in human clinical trials (including in UOB; see NCI-17-C-0086/NCT03108066: An Open-label Phase 2 Study to Evaluate PT2385 for the Treatment of Von Hippel-Lindau Disease-Associated Clear Cell Renal Cell Carcinoma; Principal Investigator: R. Srinivasan, UOB, NCI) as a benchmark for binding mode, docking simulation studies of the HTS leads were performed with ICM-Pro protein/small molecule docking software. Three lead compounds were found to bind only to the HIF2-alpha PAS B domain, and not to the HIF1-alpha protein, suggesting a mechanism of action similar to PT2385 in which the inhibitor prevents HIF2-alpha:ARNT dimerization in VHL deficient cells. Additionally, cell based assays showed the compounds to be active against key features of ccRCC oncogenesis. Interestingly, treatment of ccRCC cell lines with the three lead compounds results in significant downregulation HIF2-alpha levels, a phenomenom not seen with other inhibitors such as PT2385, and a potential mechanism to circumvent or overcome resistance to this newly targeted oncogenic signaling pathway. Further work along these lines should reveal key favorable interactions between the HIF2-alpha protein and ligand, better define target engagement and support future structure-activity relationship experiments in drug development studies.